Antisense nucleic acids are nucleic sequences capable of hybridizing selectively with target-cell messenger ribonucleic acids (mRNAs) so as to inhibit their translation into protein. These oligonucleotides form double-strand regions with the target mRNA, in a local manner, by interaction of the classic Watson-Crick type.
Many pathological states are the consequence of the expression of an abnormal gene within a cell. Such foreign genes can be integrated in the cellular deoxyribonucleic acid (DNA), for example, by a viral infection, and can therefore be expressed by the cell. The same is true for numerous oncogenes which are capable of conferring a cancerous phenotype to a eukaryotic cell, thus resulting in a tumor in an entire organism.
One approach proposed for inhibiting the action of such genes is based on the use of antisense oligonucleotides [1–3]. Indeed, the regulation of the expression of target genes by means of antisense oligonucleotides constitutes a therapeutic approach in the early stages of development. This approach depends on the capacity of the oligonucleotides to hybridize specifically at complementary regions of a nucleic acid and to thereby inhibit specifically the expression of target genes. This inhibition can take place either at the translational level by an antisense oligonucleotide or at the transcriptional level by an antigene oligonucleotide.
The therapeutic application of antisense technology has been extensively investigated in numerous viral infections, including the acquired immunodeficiency virus [4], the influenza virus [5], the Epstein-Barr virus [6], human papillomaviruses [7, 8] and the herpes simplex virus [9, 10].
It can be a question, for example, of synthetic oligonucleotides of small size, complementary to cellular mRNA, which are introduced into the target cells. Such oligonucleotides have been described, e.g., in European Patent Application No. 92 574. It can also be a question of antisense genes whose expression in the target cell generates RNA complementary of cellular mRNA. Such genes have been described, e.g., in European Patent Application No. 140 308.
Nevertheless, the in vivo use of antisense nucleic acids has encountered a number of difficulties which have, to date, limited their therapeutic exploitation.
In fact, nucleic acids exhibit a high degree of sensitivity to degradation by the enzymes of the organism, such as the nucleases [11, 12], which necessitates the use of high doses. Moreover, they exhibit weak penetration into certain cell types and an intracellular distribution which is often inadequate, both of which can render them deficient in therapeutic effect. Additionally, it is important to have available sequences that are sufficiently selective and stable so as to obtain a specific effect without altering other cell functions.
Since the first attempts by Stephenson and Zamecnik [13] to inhibit the Rous sarcoma virus using phosphodiester oligonucleotides, numerous efforts have been made to optimize the efficacy of these oligonucleotides, notably with regard to their cellular penetration [14–16], attachment to their target [17] and resistance to nucleases [18–20].
Insufficient resistance of oligonucleotides to nucleases remains a problem that limits the developmental possibilities of this therapeutic strategy. As an attempt to resolve this problem, it has been proposed to chemically modify the phosphodiester skeleton of the nucleic acids so as to create new classes of artificial oligonucleotides [21–23]. Among these modified oligonucleotides are phosphonate, phosphoramidate and phosphorothioate oligonucleotides which are described, e.g., in International Patent Application PCT No. WO 94/08003, or oligonucleotides coupled to different agents such as cholesterol, a peptide, a cationic polymer, etc.
Although certain of these modified oligonucleotides exhibit good resistance to nucleases, these modifications can have the drawback of being accompanied by the loss of other properties which are important for the antisense activity, such as their affinity for the RNA targets, their capacity to modulate the degradation of RNAs by RNases, and their power of penetration and distribution in the cell compartments remain very weak [1, 22, 24]. Furthermore, their biological activity is not always increased and they can exhibit certain secondary effects linked to the presence of non-natural motifs in their structure. In fact, the oligonucleotides modified in this manner exhibit certain undesirable characteristics such as nonspecific interactions with cellular proteins and a high level of cytotoxicity [25–29].
Another method enabling increased resistance of the oligonucleotides to nucleases but using natural phosphodiesters consists of grafting on the 3′ end of the sequence to be protected a dodecanol conjugate (European Patent Applications No. EP 117 777 and No. EP 169 787).
In order to resolve the previously mentioned drawbacks, it has also been proposed in International Patent Application PCT No. WO 94/12633, to add to one and/or both ends of the antisense sequence to be protected nucleotide sequences whose secondary structure is presented in the form of loops or hairpins, capable of preventing the nucleases from degrading the antisense sequence [30–35]. However, this technique is not satisfactory because the presence of the supplementary nucleotides of the hairpin sequences impedes the hybridization of the antisense sequence with the target nucleic acids.